Manganese-containing antibiotic agents

ABSTRACT

Manganese complexes of various monovalent and divalent polyether antibiotics are provided which act as coccidiostats and growth promoting agents when administered to food-producing animals such as cattle, sheep, swine and poultry. Soluble manganese salt is added to a fermentation beer containing the polyether antibiotics to form an insoluble, recoverable biomass containing the desired manganese antibiotic complex. This biomass, in a dried form, can be given orally to animals such as cattle, sheep, swine and poultry. 
     The subject manganese complexes include: linear monovalent and divalent polyethers (monensin, nigericin, lasalocid, lysocellin, etc.); non-glycolic monovalent monoglycoside polyethers (septamycin, dianemycin, lenoremycin, carriomycin and antibiotic A-204); mononitrogen-containing divalent pyrrole ethers (calcimycin, X-14547, etc.); polynitrogen-containing divalent pyrrole ethers (A-23187, etc.); glycolic monovalent monoglycoside polyethers (etheromycin, etc.); other polyether antibiotics including ionomycin, aabomycin, disnerycin, duamycin, BL-580, K-41, SF-1195, M-4164A, A-32887, 30,504RP, 38,986, 44,161, 47,433, 47,434 and 47,224. The above manganese complexes are also active agents for improving cardiovascular function in animals. 
     Purified manganese-containing antibiotic complexes can be extracted from the biomass using suitable organic solvents, followed by crystallization or precipitation of the purified complexes. The purified products can be administered as boluses, subcutaneous implants, or as injectable preparations.

This is a continuation of application Ser. No. 196,722 filed Oct. 8,1980 now abandoned.

BACKGROUND

This invention relates to manganese complexes of various polyetherantibiotics as new compositions of matter, processes for making thesemanganese complexes, and to processes for the administration ofmanganese complexes of polyether antibiotics to food-producing animalssuch as cattle, sheep, swine, and poultry to promote growth, to enhancefeeding efficiency, and/or to combat coccidial infections. The subjectmanganese complexes of polyether antibiotics may be administered orallyas feed additives in the crude form, or may be first purified, andadministered as boluses, parenterally as injections, or as subcutaneousimplants.

Cardiovascular function in animals may be improved by administeringmanganese complexes of polyether antibiotics. The manganese complexes ofpolyether antibiotics formed in fermentation beers as described hereinmay be purified further for pharmaceutical use in humans.

Polyether antibiotics can be generally characterized as carboxylic acidionophores which can be produced by growing Streptomyces typemicroorganisms in suitable nutrient media. These polyether antibioticshave a basic structure generally consisting essentially of the elementsoxygen, hydrogen and carbon (and sometimes nitrogen) and have amolecular weight in the range of about 300 to about 1800, most oftenfrom about 400 to about 1200. They have low solubility in water, aregenerally soluble in low molecular weight alcohols, ethers, and ketones;and have at least one, and usually one or two, carboxylic acid groups. Agenerally comprehensive review of this class of antibiotics is set forthin Westley, Adv. Appl. Microbiology 22, 177-233 (1977). As is mentionedtherein, at least twenty different polyether antibiotics were known atthe time the article was written. Since then, additional polyetherantibiotics have been discovered.

In Westley (op. cit.), the known polyether antibiotics are divided intofour separate classes based on the ability of the particular antibioticto effect the transport of monovalent and divalent cations and based onthe chemical structure of the particular antibiotic. Westley'sclassification system is adopted herein.

Westley defined Class 1a as monovalent polyether antibiotics. Inaddition, the Class 1a polyether antibiotics have a generally linearconfiguration, i.e., the carboxylic portion of the polyether molecule isattached either directly or indirectly to a terminal ring structure, andinclude about four to six tetrahydropyran and/or -furan structures, andup to six total ring structures. Class 1a includes monensin,laidlomycin, nigericin, grisorixin, salinomycin, narasin, lonomycin,X-206, SY-1, noboritomycins A and B, mutalomycin and alborixin. Class 1aantibiotics may also be described as "linear monovalent polyetherantibiotics".

According to Westley's system, monovalent monoglycoside polyetherantibiotics belong to Class 1b. These polyether antibiotics belong toClass 1b. These polyether antibiotics include a glycoside typestructure, more specifically, a2,3,6-trideoxy-4-O-methyl-D-erythrohexapyranose moiety, which isattached to the polyether molecule such that a non-linear type moleculeis formed, i.e., the carboxylic portion of the polyether molecule isattached either directly or indirectly to a non-terminal ring structureor the molecule has a side chain ring structure, e.g., a2,3,6-trideoxy-4-O-methyl-D-erythrohexapyranose moiety. The polyetherantibiotics of this class usually contain about six or seventetrahydropyran and/or -furan structures.

Class 2a antibiotics as defined by Westley are divalent polyethers, andhave a generally linear configuration. They may contain from about twoto about three tetrahydropyran and/or -furan structures, and up to aboutthree total ring structures. Nitrogen atoms are not present in the Class2a molecules. Included within Class 2a are lasalocid and lysocellin. TheClass 2a polyether antibiotics are hereinafter sometimes designated"non-nitrogen containing divalent polyether antibiotics".

Class 2b in Westley's system are divalent pyrrole polyethers. Incontrast to the other classes, the Class 2b antibiotics contain one ormore nitrogen atoms.

Lasalocid is included in Class 2a as defined by Westley. Lasalocid wasdiscovered by Julius Berger et al in media fermented with a Streptomycesmicroorganism isolated from a sample of soil collected at Hyde Park,Mass. [Cf. Berger et al, J. Am. Chem. Soc. 73, 5295-8 (1951)].Originally this material was known by the code name X-537A. About 1969lasalocid was found to possess coccidiostatic activity. Later thisactivity was established for monensin, nigericin, salinomycin, andnarasin all of which belong to Class 1a.

The polyether antibiotics have usually been recovered and employed inthe form of their sodium salts. For example, a process for recoveringlasalocid from its fermentation broth is disclosed in the Berger et alarticle (op. cit.). In this process, the antibiotic or its alkali metalsalts are extracted into various organic solvents with subsequentevaporation of the solvents in a multi-step operation.

A process for the recovery of carriomycin from fermentation beer isdescribed by Imada et al in J. Antibiotics 31, 7-14 (1978). In thedisclosed process, fermented beer containing the carriomycin antibioticwas adjusted in pH with concentrated NaOH and acetone was then added.After stirring the mixture for 1 hour at room temperature, mycelia werefiltered off and extracted again with acetone. The extracts werecombined and concentrated in a vacuum until no acetone remained. Theconcentrated aqueous solution was extracted twice with equal volumes ofethyl acetate, followed by drying with anhydrous Na₂ SO₄. The extractswere concentrated in a vacuum and passed through a column of activatedcharcoal, then the column was washed with ethyl acetate. The fractionsactive against Staphylococcus aureus FDA 209P were combined and thesolvent was evaporated. To the oily residue was added n-hexane. Theresultant solid material was collected by filtration and crystallizedfrom aqueous acetone. On recrystallization from aqueous acetone,crystals of the mixed sodium and potassium salts of carriomycin wereobtained, the mixture was dissolved in aqueous acetone, and the solutionwas extracted twice with equal volumes of ethyl acetate. The extractswere dried with anhydrous Na₂ SO₄ and concentrated to dryness in avacuum. The resultant crystalline powder was recrystallized from aqueousacetone to yield carriomycin free acid.

As is apparent from the above example, such processes can be quitecomplicated and can require the use of relatively large quantities ofvarious organic solvents, at least some of which may be quite expensive.In addition, such solvent recovery processes inevitably will sufferantibiotic yield losses as well as losses of the various organicsolvents used in the process. There is thus a continuing need forantibiotic preparation and recovery processes which effectively andefficiently produce polyether antibiotics in a form suitable for use asfeed additives.

GENERAL DESCRIPTION

Manganese complexes of polyether antibiotics can be advantageouslyformed by adding water-soluble manganese salts to the fermentation brothin which such antibiotics have been produced. When formed in afermentation beer, the formation of these complexes facilitates therecovery of the polyether antibiotics from the fermentation beer inwhich the antibiotics have been produced by, among other things,avoiding the necessity of using recovery methods which involveextractions with organic solvents followed by their subsequentpurification and reuse. The resulting broth-insoluble manganesecomplexes of the antibiotics can then be recovered from the broth andemployed, for instance, as coccidiostatic, feeding efficiency improvingand growth-promoting agents for poultry. Upon further purification, therecovered manganese complexes of these polyether manganese complexes maybe utilized in stimulating cardiovascular function in animals.

An antibiotic-containing fermentation broth can be prepared inconventional manner by fermenting a nutrient-containing liquidfermentation medium inoculated with a Streptomyces microorganism capableof producing the desired antibiotic. Suitable liquid fermentation mediaare generally aqueous dispersions containing a source of assimilablenitrogen and carbohydrates. Nitrogen sources for use in the fermentationmedia herein can include, for example, yeast, yeast-derived products,corn meal, bean meal, e.g., soy bean meal, etc. Carbohydrate sources foruse in the fermentation media herein can include, for example, sugar,molasses, corn-steep liquor and the like. The fermentation media canalso contain a variety of optional ingredients, if desired, such as forexample, pH adjustment agents, buffers, trace minerals, antifoam agents,filter aids, etc.

The antibiotic can be prepared by growing the Streptomyces microorganismin an aerated, agitated, submerged culture with the pH of the brothadjusted to about neutral, i.e., from about 6.5 to 7.5. Fermentation cangenerally be carried out at slightly elevated temperatures, e.g.,between about 25° C. and 35° C. Incubation of the broth can be carriedout for a period of several days, e.g., from about 4 to 6 days or longerif it is economically advantageous to do so.

The novel manganese complexes of the present invention can be formedfrom any of the known polyether antibiotics which include: linearmonovalent and divalent polyethers (monensin, nigericin, lasalocid,lysocellin, etc.); non-glycolic monovalent monoglycoside polyethers(septamycin, dianemycin, lenoremycin, carriomycin and antibiotic A-204);mononitrogen-containing divalent pyrrole ethers (calcimycin, X-14547,etc.); polynitrogen-containing divalent pyrrole ethers (A-23187, etc.);glycolic monovalent monoglycoside polyethers (etheromycin, etc.); otherpolyether antibiotics including ionomycin, aabomycin, disnerycin,duamycin, BL-580, K-41, SF-1195, M-4164A, A-32887, 30,504RP, 38,986,44,161, 47,433, 47,434 and 47,224.

Detailed descriptions of these antibiotics are presented in succeedingparagraphs.

DESCRIPTION OF SPECIFIC ANTIBIOTICS

A more detailed description of members of Westley's Class 1a polyetherantibiotics is given below. These antibiotics have a generally linearconfiguration. Their manganese complexes can be made as describedherein.

Monensin can be produced by inoculating the above described fermentationmedium with a Streptomyces cinnamonensis microorganism. Such amicroorganism is on unrestricted deposit under the number ATCC 15413 atthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 (hereinafter referred to as the American Type CultureCollection).

Monensin is characterized chemically as2-[5-ethyltetrahydro-5-[tetrahydro-3-methyl-5-[tetrahydro-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-2H-pyran-2-yl]-2-furyl]-2-furyl]-9-hydroxy-β-methoxy-α,γ,2,8-tetramethyl-1,6-dioxaspiro[4.5]decane-7-butyricacid. This material has the following structural formula: ##STR1##Monensin is described in greater detail in U.S. Pat. No. 3,501,568 andU.S. Pat. No. 3,794,732.

Nigericin can be produced by inoculating the fermentation medium with aStreptomyces violaceoniger microorganism. Such a microorganism is onunrestricted deposit at NRRL B1356 at the Northern Research andDevelopment Division, Agricultural Research Service, United StatesDepartment of Agriculture, Peoria, Ill. (hereinafter referred to as theAgricultural Research Service).

Nigericin is characterized chemically as a stereoisomer oftetrahydro-6-([9-methoxy-2,4,10-trimethyl-2-[tetrahydro-5-methyl-5-[tetrahydro-3-methyl-5-[tetrahydro-6-hydroxy-6-(hydroxymethyl)-3,5-dimethyl-2H-pyran-2-yl[-2-furanyl]-2-furanyl]-1,6-dioxaspiro-(4.5)dec-7-yl]-methyl-α,3-dimethyl-2H-pyran-2-aceticacid). This antibiotic has the following structural formula: ##STR2##

Nigericin is also known by the names polyetherin A, antibiotic X-464,antibiotic K178, helexin C and azolomycin M. Nigericin (and itscharacteristics and preparation) is described in greater detail in U.S.Pat. No. 3,555,150; U.S. Pat. No. 3,794,732, Harned et al, Antibioticsand Chemotherapy, Volume 1, Number 9 (December, 1951) pages 594-596;Steinrauf et al, Biochemical and Biophysical Research Communications,Volume 33, Number 1 (1968) pages 29-31 and Stempel et al, The Journal ofAntibiotics, Volume XXII, Number 8 (August, 1969) pages 384-385.

Salinomycin can be produced by inoculating a fermentation medium with aStreptomyces albus microorganism which is on deposit under number ATCC21838 at the American Type Culture Collection mentioned previously.Salinomycin was reported by Miyazaki et al, J. Antibiotics 27, 814-21(1974) as having the following structural formula: ##STR3## The abovearticle sets forth methods of preparation and properties of salinomycinand U.S. Pat. No. 3,857,948 to Tanaka et al also discloses methods forthe preparation of the salinomycin antibiotic.

Narasin (also known as 4-methylsalinomycin) can be produced byinoculating a fermentation medium with a Streptomyces aureofaciensmicroorganism which is on unrestricted deposit at the AgriculturalResearch Service mentioned previously under culture numbers NRRL 5758and 8092. The structure of narasin was reported by Berg et al, J.Antibiotics 31, 1-6 (1978) as the following: ##STR4## The antibiotic isalso the subject of U.S. Pat. Nos. 4,035,481 and 4,038,384 to Berg etal.

The antibiotics noboritomycin A and B are the fermentation products ofthe microorganism Streptomyces noboritoensis which is one deposit atAgricultural Research Service under the number NRRL 8123. A method forthe preparation of these antibiotics and their chemical structure wasreported by Keller-Juslen et al in J. Antibiotics 31, 820-828 (1978).The antibiotics have the structural formula: ##STR5## In noboritomycinA, R is methyl and in noboritomycin B, R is ethyl.

The antibiotic grisorixin is produced from the microorganismStreptomyces griseus as reported by Gachon et al, Chem. Comm., 1421-1423(1970) and J. Antibiotics 28, 345-350 (1975). As is disclosed in U.S.Pat. No. 4,161,520 to Osborne et al, the microorganism is on deposit atthe Institut National de la Recherche Agronomique where it has beenassigned the designation INRA SAB 2142. Grisorixin is structurally verysimilar to nigericin, the only difference being the presence of anadditional oxygen in nigericin. The structural formula for grisorixinis: ##STR6## Various derivatives of grisorixin are disclosed by Gachonet al, J. Antibiotics 28, 351-357 (1975).

Antibiotic X-206 was first reported by Berger et al, J. Am. Chem. Soc.73, 5295-5298 (1951) and has the following structure as reported byBlount et al, Chem. Comm., 927-928 (1971): ##STR7## Methods forpreparation of the X-206 antibiotic as well as further particulars as toits properties will be found in U.S. Pat. Nos. 3,839,557 to Raun and3,794,732 to Raun.

The antibiotic lonomycin has the following structural formula asreported by Mitani et al, J. Antibiotics 31, 750-755 (1978): ##STR8## Amethod for producing the antibiotic is given by Omura et al, J.Antibiotics 29, 15-20 (1976). The antibiotic was also identified byOshima et al, J. Antibiotics 29, 354-365 (1976) as DE-3936 and wasdetermined to be identical to emercid reported by Riche et al, J. C. S.Chem. Comm., 951-952 (1975) and to 31,559RP reported by Rhone Poulenc:Japan Patent, Kokai 50-129, 796 (Oct. 14, 1975) U.S. Pat. No. 3,950,514to Sawada et al discloses the lonomycin antibiotic as being produced bythe Streptomyces ribosidicus microorganism which has been depositedunder number ATCC 31051 at the American Type Culture Collection.

The following structural formula was determined by Gachon et al, J.Antibiotics 29, 603-610 (1976) for the antibiotic alborixin: ##STR9##Certain characteristics of the antibiotic were presented in the articleby Delhomme et al, J. Antibiotics 29, 692-695 (1976). The alborixinantibiotic is produced from a Streptomyces albus microorganism and as isdisclosed in U.S. Pat. No. 4,161,520 to Osborne et al, the microorganismis on deposit at the Institut National de la Recherche Agronomique andassigned the designation INRA SAB 3840.

Mutalomycin is produced by strain S11743/A of the Streptomyces mutabilismicroorganism which has been deposited at the Agricultural ResearchService under number NRRL 8088. A method for preparing the antibioticand its physical and chemical properties were reported by Fehr et al, J.Antibiotics 30, 903-907 (1977). The structural formula of mutalomycinis: ##STR10## As reported by Fehr et al, J. Antibiotics 32, 535-536(1979).

The antibiotic laidlomycin has been described by Kitame et al, J.Antibiotics 27, 884-887 (1974), the antibiotic being produced by theStreptomyces eurocidicus var. Asterocidicus microorganism which has beenindexed as species S-822 at the Department of Bacteriology, TohokuUniversity School of Medicine, Sendai, Japan. The chemical structure oflaidlomycin was reported by Westley, Adv. Appl. Microbiology 22, 177-223(1977) as being: ##STR11##

The laidlomycin antibiotic also appears to be the subject of U.S. Pat.No. 4,016,256 to Ishida et al.

The antibiotic SY-1 is the fermentation product of a Streptomyces albusmicroorganism, a culture of which has been deposited at the AmericanType Culture Collection under accession number ATCC 21838. As depictedin U.S. Pat. No. 4,138,496 to Shibata et al, antibiotic SY-1 has thefollowing structural formula: ##STR12## The structure of antibiotic SY-1is quite similar to that of salinomycin, the only apparent structuraldifference being that salinomycin contains a hydroxyl group on the ringdesignated "C".

Lasalocid can be prepared by inoculating the fermentation medium with aStreptomyces lasaliensis microorganism. Lyophilized tubes of thisculture bearing the laboratory designation X-537A were originallydeposited at the Agricultural Research Service under the identificationnumber NRRL 3382. Replacement has been made with a culture given theidentification number NRRL 3382R. A culture of this microorganism whichproduces lasalocid is also available from the American Type CultureCollection, Rockville, Md., under the number ATCC 31180.

The antibiotic lasalocid has been chemically identified in U.S. Pat. No.4,164,586 to Westley as6-(7(R)-[5(S)-ethyl-5-(5(R)-ethyltetrahydro-5-hydroxy-6(S)-methyl-2H-pyran-2(R)-yl)tetrahydro-3(S)-methyl-2(S)-furyl]-4(S)-hydroxy-3(R),5(S)-dimethyl-6-oxononyl)-2,3-cresoticacid. A maganese lasalocid complex, dissolved in methanol is describedby Degani, et al, Ion Binding by X-537A, Biochem. 13, 5022-33 (1974).However, a solid manganese complex of lasalocid is not disclosed, and noutility for the manganese complex of lasalocid in methanol or hexane isdisclosed. The stated purpose of the study was to determine formationconstants.

The structural formula for lasalocid is as follows: ##STR13##

A method for producing the antibiotic lysocellin was disclosed by Liu etal in U.S. Pat. No. 4,033,823. The method involves the cultivation of astrain of Streptomyces longwoodensis which is on deposit at the AmericanType Culture Collection under the designation ATCC 29251. The structureof lysocellin is as follows: ##STR14## Suitable methods for preparingthe lysocellin antibiotic are disclosed in the above-mentioned patent.The characteristics of lysocellin were first discussed in the article byEbata et al, J. Antibiotics 28, 118-121 (1975).

Additional polyether antibiotics for forming the manganese complexes ofthe subject invention include the antibiotics septamycin, dianemycin,A-204, lenoremycin and carriomycin. These latter antibiotics arenon-glycolic, monovalent monoglycoside polyethers in Westley's Class 1b.

Septamycin is also known as A-28695 and is the subject of U.S. Pat. Nos.3,839,558 and 3,839,559 to Hamill et al. As is set forth byKeller-Juslen et al, J. Antibiotics 28, 854-859 (1975), the antibiotichas the structural formula: ##STR15## The antibiotic is produced fromthe cultivation of a Streptomyces hygroscopicus microorganism which hasbeen deposited under number NRRL 5678 at the Agricultural ResearchService. The above-mentioned patents to Hamill et al classified theseptamycin producing microoganism as a Streptomyces albus microorganism,a culture of which has been deposited at the Agricultural ResearchService under accession number NRRL 3883. Further characteristics and amethod for producing the antibiotic are set forth in the article byKeller-Juslen et al mentioned above.

Dianemycin is the fermentation product of a microorganism which is astrain of Streptomyces hygroscopicus which is on unrestricted deposit asNRRL 3444 at the Agricultural Research Service. Dianemycin wascharacterized by Steinrauf et al, Biochemical and Biophysical ResearchCommunications 45, 1279-1283 (1971) as having the structure formula:##STR16## U.S. Pat. No. 3,577,531 to Gorman et al and U.S. Pat. No.3,711,605 to Hamill et al disclose the description, preparation andcharacteristics of dianemycin.

The antibiotic A-204 is described and a method for its preparationdisclosed in U.S. Pat. No. 3,705,238 to Hamill et al and in U.S. Pat.No. 3,794,732 to Raun. The term A-204 is used to designate the differentcomponents obtained by fermentation in the presence of Streptomycesalbus microorganism under aerobic conditions in a culture mediumcontaining assimilable sources of carbon, nitrogen and inorganic salts.According to U.S. Pat. No. 3,794,732 to Raun, the organism capable ofproducing antibiotic A-204 has been placed on permanent deposit, withoutrestriction, with the culture collection of the Agricultural ResearchService, and is available to the public under culture number NRRL 3384.

Component I of A-204 is the most important and the most abundant.Component II constitutes about 5% of the mixture of A-204 componentsproduced and the other components are obtained in smaller quantities.The structural formula shown below is that of the acid of A-204 I.##STR17##

The antibiotic lenoremycin is the fermentation product of a Streptomyceshygroscopicus microorganism which is deposited under number ATCC 21840at the American Type Culture Collection. The antibiotic was described byKubota et al, J. Antibiotics 28, 931-934 (1975). The structure reportedby Liu et al, J. Antibiotics 29, 21-28 (1976) is as follows: ##STR18##The Liu article also stated that lenoremycin is identical to theantibiotic A-130A described in Japanese patent publication No. 7304558of Shionogi. The antibiotic A-130A is also the subject of U.S. Pat. No.3,903,264 to Oikawa et al. The above structure is also reported inBlount et al, Chem. Comm. 853-855 (1975) who designated the antibioticas Ro 21-6150.

The antibiotic carriomycin has the following structural formula asreported by Imada et al, J. Antibiotics 31, 7-14 (1978): ##STR19## Theantibiotic is produced by strain T-42082 of the Streptomyceshygroscopicus microorganism which has been deposited at the Institutefor Fermentation, Osaka, Japan, under accession number IFO 13609 and atthe American Type Culture Collection under accession number ATCC 31080.The carriomycin antibiotic is the subject of U.S. Pat. No. 4,069,316 toImada et al.

While the above description of the various known non-glycolic polyetherantibiotics have generally identified the antibiotics as being singlecompounds, it should be recognized that at least some of these polyetherantibiotics are produced as an antibiotic complex of structurallyrelated factors containing varying proportions of each factor. As anexample, the structure for A-204 set forth previously is A-204 factor Iwhich is produced in combination with other factors in ratios dependingupon fermentation conditions. It should, therefore, be realized that thepresent invention comprehends the manganese complexes of the variousfactors of the non-glycolic polyether antibiotics whether in combinationwith other factors or in their isolated form as well as their use inpromoting growth, enhancing feeding efficiency and treating coccidialinfections in poultry, and in stimulating cardiovascular function inanimals. Furthermore, manganese complexes of derivatives of thepreviously mentioned non-glycolic polyether antibiotics are also withinthe scope of the present invention. For example, U.S. Pat. No. 3,985,872to Chamberlin is directed to dihydro A-204 and U.S. Pat. No. 3,907,832to Hamill is directed to monoether and monothioether derivatives ofA-204. Therefore, as used herein, the specific name of the polyetherantibiotic, e.g. A-204, encompasses all of the factors of theantibiotic, e.g. A-204 I and II, as well as isomers, homologs, andderivatives thereof.

For further particulars as to characteristics and methods for thepreparation of certain of the above polyether antibiotics, reference ismade to U.S. Pat. No. 3,995,027 to Gale et al and the patents citedtherein and to U.S. Pat. No. 3,794,732 to Raun and the patents andarticles cited therein.

The subclass 2b nitrogen-containing pyrrole ether antibiotics includethe antibiotic X-14547 (mononitrogen, divalent) and the antibioticA-23187 also known as calcimycin (polynitrogen-containing divalent). Thepyrrole ether antibiotic known under the code designation X-14547 ischaracterized chemically asα-(R),5(S)-dimethyl-6(R)-1-ethyl-4-[4-(R)-(2)pyrrolylcarbonyl)-1(S)-ethyl-3a(R),4,5(R),7a(R)-tetrahydroindan-5-yl]-1(E),3(E)-butadienyl-tetrahydropyran-2-acetic acid. The antibiotic isproduced by a Streptomyces sp. X-14547 microorganism, a culture of whichhas been deposited under designation number NRRL 8167 at theAgricultural Research Service. The X-14547 antibiotic has the followingstructural formula: ##STR20## Further details of the characteristics ofthe X-14547 antibiotic and processes for its production and recovery aredisclosed in U.S. Pat. No. 4,100,171 to Westley et al, in U.S. Pat. No.4,161,520 to Osborne et al, and in the articles by Liu et al, J.Antibiotics 32, 95-99 (1979) and Westley, J. Antibiotics 32, 100-107(1979).

The pyrrole ether antibiotic known under the code designation A-23187(also known as calcimycin) is the subject of U.S. Pat. No. 3,923,823 toGale et al. The patent discloses that the A-23187 antibiotic has anappreciable affinity for Cd++, moderate affinity for Ni++, Zn++, Co++and Be++, and no apparent affinity for Hg++ and suggests that because ofits preferential binding of certain cations, the antibiotic can beemployed in applications wherein the selective removal of particularcations is desired. It was reported by Pfeiffer et al, Biochemistry,Volume 15, Number 5, 935-943 (1976) that an A-23187 complex ofmanganese, as well as A-23187 complexes of other divalent cations, wasused to investigate the selectivity of the antibiotic for divalentcations over monovalent cations. See also, Pfeiffer et al, Biochemistry,Volume 13, Number 19, 4007-4014 (1974). However, there is no disclosureof a specific utility for the manganese complex of A-23187 in the abovearticle. The use of the free acid or calcium salt of the A-23187antibiotic in a method of enhancing the contractile force of themammalian heart muscle in a warm-blooded mammal is disclosed in U.S.Pat. No. 3,985,893 to Holland et al.

The A-23187 antibiotic is produced by culturing a Streptomyceschartreusis microorganism. A culture of this microorganism has beendeposited in the collection of the Agricultural Research Service underaccession number NRRL 3882. The A-23187 antibiotic has the structuralformula: ##STR21## Further details of the characteristics of theantibiotic and processes for its production and recovery are set forthin U.S. Pat. No. 3,923,823 to Gale et al.

The glycolic monovalent monoglycoside polyether antibiotics includeetheromycin (Westley Class 1b). The antibiotic etheromycin (also knownas C20-12 and CP 38295) has the chemical structure: ##STR22## Thisstructure was published by Mitani et al in J. Antibiotics 31, 750-755(1978) who also noted that etheromycin is the same as the T-40517antibiotic. According to Westley, Ad. Appl. Microbiology 22, 177-223(1977), etheromycin is produced from the Streptomyces hygroscopicusmicroorganism, a culture of which is deposited under number ATCC 31050at the American Type Culture Collection. Additional details concerningthe etheromycin antibiotic can be found in the previously mentioned U.S.Pat. No. 4,129,578 to Celmer et al.

Those polyether antibiotics for which structural information is not yetavailable, and which may be used to make the novel manganese complexesof the subject invention include ionomycin; aabomycin; disnerycin;duamycin; BL-580; K-41; SF-1195; M-4164A; A-32887; 30,504RP; 38,986;44,161; 47,433; 47,434; and 47,224. Available information about theseantibiotics is presented below.

The antibiotic ionomycin is the fermentation product of the Streptomycesconglobatus sp. nov. trejo microorganism which has been deposited underaccession number ATCC 31005 at the American Type Culture Collection. Theantibiotic has been characterized by Liu et al, J. Antibiotics 31,815-819 (1978) which also exhibits a suitable method for the preparationof the antibiotic. The ionomycin antibiotic is also the subject of U.S.Pat. No. 3,873,693 to Meyers et al.

The isolation and characterization of the polyether antibiotic K-41 wasreported by Tsuji et al, J. Antibiotics 29, 10-14 (1976). The antibioticis produced from a strain of Streptomyces hygroscopicus microorganismdeposited at the Fermentation Research Institute, Chiba, Japan, withdeposit number FERM-P 1342. The above article reports that theantibiotic is the subject of Japanese Patent No. 49-14692 (1974). Amethod utilizing the antibiotic K-41 in protecting plants from mites isdisclosed in U.S. Pat. No. 4,148,881 to Ishiguro.

U.S. Pat. No. 3,812,249 to J. H. E. J. Martin et al is directed to thepolyether antibiotics BL-580 α and β. These antibiotics are products ofa Streptomyces hygroscopicus microorganism which has been deposited atthe Agricultural Research Service under deposit number NRRL 5647. Theabove-mentioned patent discloses suitable methods for the preparation ofthe BL-580 antibiotic. U.S. Pat. No. 4,132,779 to Hertz et al disclosesthe antibiotic BL-580 zeta which is produced by a mutant strain ofStreptomyces hygroscopicus derived by treatment of a natural section,single colony isolate of Streptomyces hygroscopicus NRRL 5647 withN-methyl-N'-nitro-N"-nitrosoguanidine. A culture of the mutant strainhas been deposited at the Agriculture Research Service under accessionnumber NRRL 11108.

The polyether antibiotic aabomycin X was recently reported in theSupplement to "Index of Antibiotics from Actinomyces" by Dr. HamaoUmezawa, J. Antibiotics 32, 79-51 (1979) and is also apparently thesubject of Japan Kokai No. 77-90697 filed July 30, 1977, in the name ofShibata, et al. The antibiotic is produced by the fermentation of themicroorganism Streptomyces hygroscopicus subsp. aabomyceticus 325-17which has been deposited at the American Type Culture Collection underdeposit number ATCC 21449. The microorganism produces an antibioticmixture which includes the factors aabomycin X and aabomycin A. Theantibiotic aabomycin A is the subject of U.S. Pat. No. 3,657,422 toMisato et al.

The antibiotic duamycin was described in Japanese Pat. No. 26719 (1970)to Kaken-Kagaku, the patent being abstracted in Chemical Abstracts 74,21895p (1971). The antibiotic SF-1195 was disclosed in Japanese Pat. No.49-132212 (1974) to Sawada et al. Disnerycin was mentioned in U.S. Pat.No. 4,159,322 to Cloyd as being a polycyclic ether antibiotic of thesame class as monensin, nigericin, grisorixin, salinomycin, narasin andlasalocid. The antibiotic identified as M-4164A was described in JapanKokai Pat. No. 50-12294 (1975) to Toyama et al.

The polyether antibiotic designated as Compound 38,986 is disclosed inU.S. Pat. Nos. 4,022,885 and 4,048,304 to Celmer et al. The antibioticis the product of a Streptomyces flaveolus microorganism, a culture ofwhich has been deposited in the American Type Culture Collection andgiven designation ATCC 31100.

The polyether antibiotic Compound 44,161 is produced by cultivating astrain of Dactylosporangium salmoneum Routien sp. nov., cultures ofwhich have been deposited at the American Type Culture Collection underaccession numbers ATCC 31222, 31223 and 31224. Additional detailsregarding this antibiotic are contained in U.S. Pat. No. 4,081,532 toCelmer et al.

The antibiotic A-32887 is the subject of U.S. Pat. Nos. 4,132,778 and4,133,876 to Hamill et al. As is described in these patents, the A-32887antibiotic is closely related to the K-41 antibiotic and is produced byculturing a strain of Streptomyces albus which has been deposited underdesignation NRRL 11109 at the Agricultural Research Service.

The two polyether antibiotics disclosed in U.S. Pat. No. 4,148,882 toCelmer et al were given the designations Compounds 47,433 and 47,434.These antibiotics are produced by a species of Actinomadura macer Huangsp. nov., a culture of which has been deposited at the American TypeCulture Collection and given the designation number ATCC 31286.

U.S. Pat. No. 3,989,820 to Florent et al is directed to the antibiotic30,504RP which is produced by culturing a microorganism calledStreptomyces gallinarius DS 25881, a culture of which has been depositedat the Agricultural Research Service under number NRRL 5785.

The polyether antibiotic given the designation Compound 47,224 isproduced by a strain of a Streptomyces hygroscopicus microorganism. Asis disclosed in U.S. Pat. No. 4,150,152 to Celmer et al, themicroorganism strain capable of producing Compound 47,224 has beendeposited at the American Type Culture Collection with the accessionnumber ATCC 31337.

While the above descriptions of the various known polyether antibioticshave generally identified the antibiotics as being single compounds, itshould be recognized that at least some of the polyether antibiotics areproduced as an antibiotic complex of structurally related factorscontaining varying proportions of each factor. As an example, thestructure for lasalocid set forth previously is lasalocid factor A whichis produced in combination with factors B, C, D and E in ratiosdepending upon fermentation conditions. Homologs of lasalocid A aredisclosed in U.S. Pat. No. 4,168,272 to Westley. An isomeric form oflasalocid is also known from U.S. Pat. No. 3,944,573 to Westley. Inaddition, monensin is produced with factors B and C as reported byWestley, Adv. Appl. Microbiology 22, 200 (1977) and narasin is producedwith factors A, B and D as is set forth in U.S. Pat. No. 4,038,384 toBerg et al. It should, therefore, be realized that the present inventioncomprehends the manganese complexes of the various factors of thepolyether antibiotics whether in combination with other factors or intheir isolated form as well as their use in promoting growth, enhancingfeeding efficiency and treating coccidial infections in poultry, and instimulating cardiovascular function in animals.

Furthermore, manganese complexes of derivatives of the previouslymentioned polyether antibiotics are also within the scope of the presentinvention. For example, various derivatives of the lasalocid antibioticare known from U.S. Pat. No. 3,715,372 to Stempel et al. In addition,derivatives of monensin are disclosed in U.S. Pat. No. 3,932,619 toBrannon et al which is directed to a metabolite produced from monensin,U.S. Pat. No. 3,832,258 to Chamberlin which is directed to thedeshydroxymethyl derivative of monensin and U.S. Pat. Nos. 4,141,907 and4,174,404 to Nakatsukasa et al are directed to deoxynarasin. Therefore,as used herein, the specific name of the polyether antibiotic, e.g.lasalocid, encompasses all of the factors of the antibiotic, e.g.lasalocid A, B, C, D and E, as well as isomers thereof, e.g.iso-lasalocid, and derivatives thereof.

For further particulars as to characteristics and methods for thepreparation of certain of the above polyether antibiotics, reference ismade to U.S. Pat. No. 3,995,027 to Gale et al and the patents citedtherein and to U.S. Pat. No. 3,794,732 to Raun and the patents andarticles cited therein.

It is also within the scope of the present invention that the novelmanganese complexes of the polyether antibiotics described herein can beused in conjunction with other active ingredients which are also usefulfor challenging coccidial infections in poultry and/or for promotinggrowth and enhancing feed efficiency in poultry. For example, themanganese complexes of polyether antibiotics may have an enhanced effectwhen used in combination with metichlorpindol. The use ofmetichlorpindol with monensin for treatment of poultry coccidiosis isdescribed in U.S. Pat. No. 4,061,755 to McDougald. Compositionscontaining certain designated polyether antibiotics and a pleuromutilinderivative which are useful in treating poultry coccidiosis aredisclosed in U.S. Pat. No. 4,148,890 to Czok et al.

To the extent necessary, the above-mentioned patents and literaturearticles mentioned in describing the various known polyether antibioticsand their uses are incorporated herein by reference.

GENERAL DESCRIPTION OF SYNTHESIS

In accordance with the present invention, the polyether antibiotic,generally in the form of its alkali metal, alkaline earth metal orammonium salt, is treated in situ in the fermentation broth or beer byadding to the antiobiotic containing broth a water-soluble manganesesalt. Addition of such a water-soluble manganese salt promotes theformation of a manganese complex of the polyether antibiotic. Such amanganese complex of the antibiotic, along with manganese complexesformed with residual nitrogen-containing compounds in the broth such asamino acids, polypeptides, and proteins, are insoluble in thefermentation broth liquid.

The manganese ions from the added manganese salt apparently formcoordination bonds with the oxygen atoms of the sparingly solublepolyether antibiotic. For example, the structure of the manganesecomplex of lasalocid is believed to be represented by the following:##STR23##

On the basis of the formation constants with ligands such as citricacid, lactic acid and tartaric acid, it is believed that manganese ionsform stronger bonds with oxygen-containing compounds than do ions suchas Mg++, Ca++, Ba++, Na+ and K+.

The manganese salt added to the fermentation broth can be chosen fromvarious water-soluble salts which ionize in the fermentation broth. Suchsalts include, for example, manganese chloride, manganese nitrate,manganese acetate, etc. Water-soluble manganese salts are generallythose which can be dissolved to the extent of about 1 percent by weightor more in water at 20° C. For maximum production of the desiredmanganese complexes, the water-soluble manganese salt should be added tothe fermented broth in an amount which is sufficient to fillsubstantially all of the possible manganese coordination sites of theproteins, polypeptides, amino acids and related compounds, in additionto substantially all of the available coordination sites of theantibiotic present. This is necessary because in general, nitrogen atomsin the polypeptides, amino acids, etc., form stronger coordination bondswith manganese than do the oxygen atoms in the polyether antibiotic.Generally, therefore, manganese salt is added to the fermentation brothin an amount sufficient to provide a manganese content of from about 3to 12 percent, and preferably, from about 5 to 10 percent by weight ofthe dried precipitate recovered from the fermentation broth ashereinafter more fully described.

The amount of soluble manganese salt to be added will depend on theamount of nutrients added to the fermentation broth during the course ofthe fermentation. The actual amount of soluble manganese salt to beadded to the broth obtained from a given mash bill can be determined bysimple laboratory precipitations followed by manganese analyses on thedried precipitates. When, for example, the preferred manganese chloridesalt is employed to form the desired manganese antibiotic complex,advantageously from about 8 to 18 gallons of a 43.5 weight percentmanganese chloride solution (sp. gr. 1.499), can be added to 1000gallons of fermentation broth.

To form the manganese antibiotic complex in the fermentation broth, pHof the broth is advantageously adjusted to about 6.5 to 7.5, andpreferably, to about 6.8 to 7.2 after addition of the soluble manganesesalt to the fermentation broth.

The insoluble manganese complexes formed upon addition of manganese saltcan be readily separated from the fermentation broth or beer byconventional filtration or centrifugation techniques. In this manner, awet biomass containing the manganese antibiotic complex is realized.This wet biomass is resistant to wild fermentations because of itsrelatively high manganese content. The wet biomass so obtained is easilydried by spray drying or drum drying procedure, and this manganeseantibiotic-containing dried product can then be used as a feed additiveper se. If the antibiotic content of the fermentation beer is lower thandesired after completion of the fermentation, crude antibiotic in itssodium salt form can be added to the fermentation beer prior to theaddition of the soluble manganese salt. In this manner, the antibioticcontent of the biomass composition to be separated from the broth can beincreased. To be suitable as a feed additive, the dried biomasspreferably contains at least about 5 percent by weight of the manganeseantibiotic complex, advantageously from about 10 percent to 50 percentby weight of the manganese antibiotic complex.

Recovery of the manganese antibiotic complexes of the present inventionin the manner described herein provides several important advantagesover known antibiotic preparation and recovery processes. The presentprocess, for example, provides a means for recovering relatively highyields of antibiotic in a salable feed additive product. Further, theuse of expensive extraction solvents and the cost associated with theprocess losses of such solvents are avoided. The present process alsopermits recovery of salable feed values present in the mycelium of theStreptomyces microorganism used to produce the antibiotic. The presentprocess further reduces the cost of waste disposal operations needed inprevious processes to deal with the mycelial mat produced duringfermentation. Use of this mat as part of the feed additive product, infact, reduces the cost of the carrier for the antibiotic material beingmarketed.

The dried, antibiotic-containing biomass recovered from the fermentationbroth as hereinbefore described can be added to conventional poultryfeed compositions as a coccidiostatic and growth-promoting agent. Suchfeed compositions generally contain whole or ground cereal or cerealbyproducts as an essential nutrient. The feed compositions can alsocontain such optional additional materials as animal byproducts, e.g.,bone meal, fish meal, etc., carbohydrates, vitamins, minerals and thelike. The manganese antibiotic complexes of the present invention aregenerally employed in the feed compositions to the extent of from about50 grams per ton to 200 grams per ton, preferably from about 75 gramsper ton to 125 grams per ton.

As was mentioned previously, the manganese complexes of polyetherantibiotic according to the present invention may also be utilized forthe stimulation of cardiovascular functions and particularly in thetreatment of ailments such as cardiogenic shock, septic shock andcongestive heart failure. Preferably, the manganese complexes areutilized for these purposes in a purified form and are administeredeither orally or parenterally to a patient requiring treatment. Oraladministration is particularly preferred for long term treatment ofchronic diseases such as congestive heart failure while parenteraladministration is preferred for emergency treatment such as in thetreatment of shock and of acute heart failure.

Purification of the manganese complexes of the present invention so thatthe complexes are more suitable for administration to humans can beaccomplished in a variety of manners. A presently preferred method forpurification of the manganese complexes from the recovered feed grademanganese complex includes the steps of, after treatment of thefermentation beer with a soluble manganese salt, acidifying the waterslurry of the manganese complex with strong mineral acid such assulfuric acid to produce a relatively low pH, e.g. a pH below about 4,preferably about 2 to about 3, and then extracting the acid form of thepolyether antibiotic from the slurry into a substantiallywater-insoluble organic solvent such as butyl acetate.

Thereafter, a lower aliphatic alcohol such as methanol is added to theorganic solvent containing the polyether antibiotic. The volume ofalcohol added is generally less than or about equal to the volume oforganic solvent, preferably about 0.25 to about 1.0 volumes alcohol toabout 1.0 volume of organic solvent. A soluble manganese salt such asmanganese chloride dissolved in the same lower aliphatic alcohol is thenslowly added with vigorous agitation to the organic solvent-alcoholmixture containing the polyether antibiotic. Preferably, about 0.5 to1.0 volumes of the alcohol containing the manganese salt are added pervolume of mixture. The amount of manganese salt added should besufficient to convert essentially all of the contained antibiotic to itsmanganese complex form. The formed manganese complexes are then filteredfrom the mixture, thoroughly washed and dried.

If greater purification of the manganese complex is desired, the aboveprocedure can be modified to include further purification steps. Onesuch modification is, prior to the addition of the lower aliphaticalcohol, adding an aqueous solution containing an alkali metal hydroxidesuch as potassium or sodium hydroxide to the organic solvent containingthe polyether antibiotic so that the antibiotic is extracted into theaqueous solution. The antibiotic is then re-extracted into the sameorganic solvent or a different water-insoluble organic solvent such asmethyl tertiary-butyl ether after acidification. These steps of themodified procedure can be repeated as many times as desired until theproper degree of purification is achieved. Thereafter, the polyetherantibiotic is contacted with the lower aliphatic alcohol and thepreviously mentioned procedure continued so as to yield the purifiedmanganese complex of the polyether antibiotic.

In the above description of the purification procedure and modificationthereof, the amount of each of the media, i.e., the organic solvent,aliphatic alcohol, aqueous solution, etc., relative to the others whenconducting the procedure may vary considerably, the primaryconsiderations being that sufficient media be utilized to obtain asatisfactory yield of the manganese complex balanced against the cost ofthe media and the capacity of the available equipment. Generally, theamount of a particular medium used to treat another medium in any of thesteps of the above procedure is about 0.1 to 10 volumes, preferablyabout 0.5 to about 5 volumes, for each volume treated.

Certain advantages are realized by the above procedure where thepurified manganese complexes are recovered from the feed grade complexesas opposed to recovery of the purified complexes from virgin mycelia.Among others, the feed grade complexes are filtered relatively easilyfrom the fermentation beer whereas filtering of virgin mycelia is veryslow and thus time-consuming. In addition, the feed grade complexes tendto be more concentrated and thus less organic solvent is required inconducting the purification procedure and volume loss of solvent will bereduced.

DESCRIPTION OF USES

The manganese complexes of the present invention may be formulated withconventional inert pharmaceutical carrier or adjuvant materials intodosage forms which are suitable for oral or parenteral administration tostimulate cardiovascular function. Such dosage forms include tablets,suspensions, solutions, hard or soft capsules, dragees and the like. Theselection of suitable materials which may be used in formulating theactive manganese complexes into oral and parenteral dosage forms will beapparent to persons skilled in the art. Such materials, either inorganicor organic in nature, should be of pharmaceutically acceptable quality,free from deleterious impurities and may include, for example, water,dimethylsulfoxide, gelatin albumin, lactose, starch, magnesium stearate,preservatives, stabilizers, wetting agents, emulsifying agents, saltsfor altering osmotic pressure, buffers, etc. which can be incorporated,if desired, into such formulations.

The quantity of manganese complex which may be present in any of theabove described dosage forms generally varies from 10 to 100 mg per unitdosage. The dosage administered to a particular patient is variable,depending upon the clinician's judgment using the criteria of thecondition and the size of the patient, the potency of the manganesecomplex and the patient's particular response thereto. An effectivedosage amount of the manganese complex can, therefore, only bedetermined by the clinician utilizing his best judgment on the patient'sbehalf. Generally, parenteral does should be from about 20 mg to about50 mg for the average size person. Smaller persons or larger persons mayrequire adjustments due to body size. Oral doses, usually capsules, buttablets can be used, generally contain about twice the parenteral dose.The frequency of the administration of the manganese complex dependsgenerally upon the patient's condition and the desired response from thepatient. Chronically ill patients may require administration every 2 to3 hours or once a day, depending on the severity of the disease and thepatient's particular response to treatment. Emergency patients generallyrequire only one dose of the manganese complex, particularly thosepatients in shock.

When administered to a patient requiring treatment, the manganesecomplexes of the present invention generally have a positive inotropiceffect with little or no chronotropic effects and display minimal, ifany, adrenergic action, have a rapid onset of action, require a smalleffective dose, are non-toxic at the effective doses, have asatisfactory duration of action, display a return to the originalpredrug values of cardiovascular activity, and exhibit continuedgenerally identical responses to subsequently repeated identicaldosages.

Illustrated in the following examples are preparation and recoverymethods for the manganese complexes of polyether antibiotics as well asfeed and feed additive compositions including these manganese complexesand their usefulness as coccidiostats and growth-promoting agents forfood-producing animals such as cattle, sheep, swine and poultry, and, inaddition, pharmaceutical compositions including these manganesecomplexes and their usefulness in stimulating cardiovascular function.These examples are in no way to be considered limiting of the presentinvention to compositions, ingredients, and processes involving thatparticular material.

EXAMPLE I A. Fermentation

About 450 ml of inoculum of Streptomyces lasaliensis culture No. NRRL3382R, obtained from the Agricultural Research Service is introducedinto 9,000 ml of fermentation medium of the following composition:

    ______________________________________                                        Soybean Flour          2%                                                     Brown Sugar            2%                                                     Corn Steep Liquor      0.5%                                                   K.sub.2 HPO.sub.4      0.1%                                                   Hodag Antifoam K-67    0.05%                                                  Water                  Balance                                                                       100.00%                                                ______________________________________                                    

The fermentation is conducted in a 20-liter, stainless steel fermentorusing the conditions listed below.

1. Amount of medium--9.45 liters.

2. Temperature--28° C.

3. Air Flow--9.0 liters per minute.

4. Mechanical agitation--One 13-cm diameter impeller rotating at 600RPM.

5. Back pressure--about 16.7 psig.

6. Time of fermentation--72 hours.

At the end of the fermentation the lasalocid assay of the beer is 1.5 gper liter.

B. Recovery

Since the assay of the beer for lasalocid is low compared to assayscommonly obtained for antibiotics, the beer is spiked with crudelasalocid which has been obtained by extracting with butyl acetate acommercial product containing approximately 81 grams of sodium lasalocidper pound.

Twenty-five grams of crude sodium lasalocid (78.5% lasalocid) dissolvedin 150 ml of methanol are added to 2000 ml of beer under constantagitation. After thorough agitation, 12.5 ml of a manganese chloridesolution (0.25 g Mn per ml) are slowly added with agitation to thefermented beer. The pH is adjusted to a value in the range 7.0-7.4.

After the treated beer has been agitated for about 30 minutes it isfiltered, without filter aid, on a Buckner funnel using No. 1 Whatmanfilter paper. The filtration proceeds rapidly to give a firm cake whichis dried in an oven. The final dried product weighs 57 grams and has anassay of 32.7% lasalocid.

The calculated recovery from beer to dried product is 82.5% derived fromthe following formula. ##EQU1##

EXAMPLE II Objective

To determine the efficacy of the new manganese lasalocid complex as ananticoccidial compound for poultry, manganese lasalocid is tested incomparison with Coban (monensin sodium) in chickens which are challengedby Eimeria tenella.

    __________________________________________________________________________                      Total                                                                              24 birds/                                                                          Initial                                                                           14     Hubbard White Initial                  Test Animals:                                                                         Species:                                                                            Avian                                                                             Number:                                                                            treatment                                                                          Age:                                                                              days                                                                             Breed:                                                                            Mountain                                                                              Sex:                                                                             Male                                                                             Weight:                                                                             330                __________________________________________________________________________                                                               g              

Test Materials

Manganese lasalocid--32.7% pure by weight

Coban (monensin sodium)--Lot No. X31211

Test Procedure

1. At 14 days of age chicks are weighed and assigned to groups.Immediately after groups are formed (composed of 12 chicks each) eachare started on their respective medicated feed ration. Each treatmentgroup is replicated twice for a total of 24 birds per group.

2. Seventy-two hours after the initiation of medication, birds areorally inoculated with approximately 100,000 Eimeria tenella oocystssuspended in a 1 cc dose.

3. Controls consist of an infected non-medicated group, a non-infectedgroup and an infected group treated with Coban.

4. Criteria for evaluation are

a. Morbidity (4th-6th day)

b. Mortality (4th-7th day)

c. Incidence of bloddy droppings (4th-6th day)

d. Body weight gain

e. Feed per gain

f. Postmortem lesions

Treatment Groups

    ______________________________________                                        Pen           Treatment                                                       ______________________________________                                        4, 9          Manganese lasalocid, 75 g/ton                                   1, 6          Manganese lasalocid, 113 g/ton                                  5, 12         Manganese lasalocid, 150 g/ton                                  7, 11         Coban, 110 g/ton                                                3, 8          Non-inoculated control                                          2, 10         Inoculated control                                              ______________________________________                                    

Rations

Rations employed in the Example II testing are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Chick Starter (corn)                                                          ______________________________________                                        % Protein    23.0      % Calcium      .98                                     M.E. Kilocalories/lb 1376*                                                                       % Total phosphorus                                                                           .89                                         Ground yellow corn    55.0                                                    Soybean meal 44%      29.0                                                    Fish solubles         2.0                                                     Meat and bone         5.0                                                     Dehydrated alfalfa meal                                                                             1.2                                                     Dried whey            1.0                                                     Animal tallow         4.0                                                     Dicalcium phosphate   1.0                                                     Hubbard super-13      0.8                                                     Vitamin and trace mineral premix                                                                    0.5                                                     Salt                  0.5                                                                           100.0                                                   ______________________________________                                         *M.E.  Metabolizable Energy                                              

Test Results

Results obtainable from the Example II testing demonstrate that asatisfactory challenge is obtained with E. tenella and that all threelevels of manganese lasalocid demonstrate excellent activity againstthis organism. In fact the birds receiving the two lower levels ofmanganese lasalocid also show superiority over the controls in weightgain and in feed efficiency. Furthermore, manganese lasalocid givesresults superior to those obtained with Coban in controlling E. tenella,in weight gain and in feed efficiency.

EXAMPLE III

An experiment is run to confirm the indication in Example II thatmanganese lasalocid has growth promoting properties for chickens.

Objective

To determine the efficacy of manganese lasalocid for promoting thegrowth and improving feed efficiency in broiler chicks.

    __________________________________________________________________________                                        Hubbard                                   Test          Total                                                                              60 birds/                                                                          Initial     White      Initial Duration                                                                           13                Animals:                                                                           Species:                                                                           Avian                                                                             Number:                                                                            treatment                                                                          Age:                                                                              2 days                                                                            Breed:                                                                            Mountain                                                                           Sex:                                                                             Male                                                                             Weight:                                                                            43 g                                                                             of                                                                                 days:             __________________________________________________________________________

Two-day old broiler type chicks are placed into Petersime starterbatteries and given feed and water ad libitum for the duration of thetest. Chicks are divided into four treatment groups which are replicatedsix times with ten chicks (males) in each replication. The test periodis 13 days. Pen live body weights are taken at 2, 7 and 14 days of age.Pen feed efficiency measurements are taken at 14 days of age.

Rations

Rations employed in the testing are summarized in Table 2.

                  TABLE 2                                                         ______________________________________                                        Chick Starter (Rye)                                                           ______________________________________                                        % Protein    23.2      % Calcium      0.98                                    M.E. Kilocalories/lb 1260*                                                                       % Total phosphorus                                                                           0.89                                        Ground rye               55.0                                                 Soybean meal 44%         29.0                                                 Fish solubles            2.0                                                  Meat and bone meal       5.0                                                  Dehydrated alfalfa meal  1.2                                                  Dried whey               1.0                                                  Animal tallow            4.0                                                  Dicalcium phosphate      1.0                                                  Hubbard Super-13 mineral 0.8                                                  Vitamin and trace mineral premix                                                                       0.5                                                  Salt                     0.5                                                                           100    lbs                                           ______________________________________                                         *M.E.  Metabolizable Energy                                              

Test Results

Results obtainable from the Example III testing demonstrate that, atlevels of 50 and 100 grams perton, manganese lasalocid greatly improvesgrowth response and feed efficiency of young broiler chicks.

EXAMPLE IV Objective

To compare efficacy of manganese lasalocid against lasalocid and zincbacitracin for promoting growth and improving feed efficiency in thechick.

    __________________________________________________________________________                                        Hubbard                                   Test          Total                                                                              60 birds/                                                                          Initial     White      Initial Duration                                                                           14                Animals:                                                                           Species:                                                                           Avian                                                                             Number:                                                                            treatment                                                                          Age:                                                                              2 days                                                                            Breed:                                                                            Mountain                                                                           Sex:                                                                             Male                                                                             Weight:                                                                            36 g                                                                             of                                                                                 days:             __________________________________________________________________________

Test Procedure

Two-day old broiler type chicks are placed into Petersime starterbatteries and given feed and water ad libitum for the duration of thetest. Chicks are randomly divided into four treatment groups which arereplicated six times with ten chicks (males) in each replication. Thetest period was 14 days. Pen live body weights are taken at 2, 7 and 15days of age. Pen feed efficiency measurements are taken at 15 days ofage.

Treatment Groups

    ______________________________________                                        Pen       Treatment          Lot No.                                          ______________________________________                                        241, 246, 249                                                                           Control                                                             256, 259, 264                                                                 245, 248, 251                                                                           Lasalocid.sup.1, 50 g/ton                                                                        P-446 E                                          254, 255, 262                                                                 244, 247, 250                                                                           Zinc bacitracin.sup.2, 50 g/ton                                                                  11207902                                         253, 258, 263                                                                 242, 243, 252                                                                           Manganese lasalocid.sup.3, 50 g/ton                                                              Prepared by                                      257, 260, 261                Procedure of                                                                  Example I                                        ______________________________________                                         .sup.1 Lasalocid  68 g/lb (Avatec)                                            .sup.2 BACIFERM10                                                             .sup.3 Manganese lasalocid  32.7% lasalocid by wt.                       

Rations

The composition of the chick starter ration is given in Table 2 ofExample III.

Test Results

Results obtainable from the Example IV testing show that manganeselasalocid fed at 50 g/ton will produce about a 45% increase in bodyweight gain compared to the control group. Under these same testconditions, lasalocid actually depressed body weight gain by about 5%compared to the control. Manganese lasalocid will produce a Feed/Grainratio of about 1.48, or an 8.6% increase over the control, whereaslasalocid, under the same test conditions, produced a Feed/Gain ratio of1.61 which was only 0.4% over the control.

EXAMPLE V

Administration of manganese lasalocid growth-promoting agent to cattlevia cattle feed composition is illustrated by this example. A cattlefeed formulation having the following composition is prepared:

    ______________________________________                                        Composition        Concentration                                              ______________________________________                                        Cracked Corn       68.5%                                                      Alfalfa Meal        5.0%                                                      Ground Cobs        10.0%                                                      Soybean Meal (50% protein)                                                                       15.0%                                                      Mineral Mixture     1.0%                                                      Salt                0.5%                                                                         100.0%                                                     ______________________________________                                    

To such a composition is added enough of the manganeselasalocid-containing dried product of Example I to provide a feedcomposition containing about 100 grams of manganese lasalocid per ton offeed composition.

The manganese lasalocid-containing feed composition is fed to cattle inamounts sufficient to provide from about 5 to 100 ppm of manganeselasalocid in the rumen fluid. Administration of the manganese lasalocidmaterial in this manner serves to promote cattle growth by enhancing theefficiency with which the cattle so treated utilize their feed.

EXAMPLE VI

The tendency of manganese lasalocid antibiotic to desirably affectacetate/propionate ratios in rumen fluid from cattle is demonstrated bymeans of an in vitro rumen fluid analysis procedure. Rumen fluid isobtained from a steer which has a surgically installed fistula openinginto the rumen. The steer is maintained on a grain diet consisting ofthe feed composition set forth in Example V. A sample of rumen fluid isstrained through four layers of cheesecloth and the eluate collected. Anequal amount of buffer solution with a pH of 7 is added to the rumenfluid. Ten ml of the diluted rumen fluid is placed in flasks with 500 mgof the same feed shown above which has been finely ground. Each ofmaterials to be tested is weighted into a separate test flask. Fourcondtrol flasks are also employed. All of the test flasks are incubatedfor 24 hours at 39° C. At the end of incubation, a pH is measured andone drop of mercuric chloride is added to each flask. The samples arecentrifuged at 3000×g for 15 minutes and the supernatant is analyzed bygas chromatographic methods for volatile fatty acids.

Analyses for acetate, propionate and butyrate compounds are performed.The results are statistically compared with the results of the analysesof the control flasks. The acetic/propionic ratios are calculated foreach treatment. Treatments with propionate production significantlyhigher than the control are evidenced in this ratio expression by lessernumbers. These treatments are then regarded as active treatments.Results of these tests are set forth in Table I immediately below.

                  TABLE I                                                         ______________________________________                                        Effect of Manganese Lasalocid on Acetate/Propionate                           Ratios of In Vitro Ruminal Fluid                                                                Positive Control                                                       Negative                                                                             Monensin  Lasalocid, ppm                                    Item*        Control  5 ppm     1    5   10                                   ______________________________________                                        Acetate/propionate                                                                         1.47     1.04      1.10 .99 0.95                                 ______________________________________                                         *Means of seven experiments, 3 reps/treatment                            

The data in Table I demonstrates that the presence of manganeselasalocid in the rumen fluid can beneficially increase the production ofpropionate within the rumen relative to acetate production. Cattlewherein such a propionate increase occurs are more efficiently able toutilize their feed in the production of meat and milk.

EXAMPLE VII

The manganese complex of lasalocid is purified by a purificationprocess, incorporated into a pharmaceutical composition, and is thenadministered to dogs to stimulate their cardiovascular function.

Purification

To a liter of fermentation beer slurry containing manganese lasalocidwhich was produced in a manner as set forth in Example I, sufficientconcentrated sulfuric acid is added to acidify the slurry offermentation beer to a pH of about 3.0. The slurry is then mixed withabout one liter of a butyl acetate organic solvent so that the manganeselasalocid is extracted in the solvent. The organic solvent whichcontains the lasalocid antibiotic is then separated from the acidicaqueous beer and is mixed with about one liter of an aqueous solution ofsodium hydroxide having a pH of about 9.0 so that the lasalocidantibiotic will be extracted into the aqueous alkaline solution. Uponseparation of the aqueous alkaline solution from the organic solvent,about one liter of methyl tertiary-butyl ether solvent is added to theaqueous solution to re-extract the lasalocid antibiotic into thesolvent. Thereafter, about 0.5 liter of methanol is first added to thesolvent and then about 0.5 liter of a solution of manganese chloride inmethanol is slowly added with vigorous agitation. A manganese complex oflasalocid is thereby formed in the methanol-solvent mixture which issubsequently filtered from the mixture, thoroughly washed withadditional methanol and then dried. The formed manganese complex oflasalocid is suitable for administration to stimulate cardiovascularfunction.

Pharmaceutical Preparation

A pharmaceutical composition containing the manganese complex oflasalocid is prepared, the composition being suitable for parenteraladministration.

The following ingredients are utilized to prepare a 5 ml parenteralsolution:

Manganese complex of lasalocid: 50 mg

Propylene glycol: 2.5 ml

Benzyl alcohol: 0.075 ml

Ethyl alcohol: 0.5 ml

Water: Balance.

Treatment

The above parenteral composition, or any other form of the manganesecomplexes of the present invention, is administered to animals, e.g.mammals such as, for instance, dogs, prophylactically for, or having acardiovascular malfunction to stimulate their respective cardiovascularfunctions. The procedure utilized is similar to that set forth in U.S.Pat. No. 4,058,620 to Westley. The electrophysical and hemodynamicresponses of the dogs are measured before and at various time intervalsafter intravenous injections of the composition. The parameters measuredare myocardial force of contraction, heart rate and blood pressure.Positive inotropic effects are sought with minimal chronotropic effectsbeing manifested in the treated animal. PG,46

EXAMPLE VIII

A manganese complex of lasalocid as purified by the procedure of ExampleVII is formulated into pharmaceutical tablets suitable for oraladministration in stimulating cardiovascular function.

Each tablet has the following composition:

Manganese complex of lasalocid: 25 mg

Lactose: 113.5 mg

Corn starch: 55.5 mg

Pregelatinized corn starch: 8 mg

Calcium stearate: 3 mg

The tablets are made by thoroughly mixing the manganese complex,lactose, corn starch and pregelatinized corn starch, passing the mixturethrough a comminuting machine and then moistening the mixture with waterin a mixer to produce a paste. The formed paste is screened to formgranules and then dried. Calcium stearate is mixed with the driedgranules and the granules compressed into tablets using a conventionaltableting machine.

EXAMPLES IX-XLV

Other manganese complexes of polyether antibiotics are produced andrecovered and the resultant complexes are used as coccidiostats andgrowth-promoting agents in food-producing animals such as cattle, sheep,swine and poultry and as pharmaceutical formulations for myocardialstimulation in animals. The antibiotics utilized in the examples aremonensin, nigericin, salinomycin, narasin, noboritomycin A and B,lysocellin, grisorixin, X-206, lonomycin, laidlomycin, SY-1,mutalomycin, alborixin, carriomycin, septamycin, dianemycin, A-204,Lenoremycin, calcimycin, X-14547, A-23187, etheromycin, ionomycin,aabomycin, disnerycin, duamycin, BL-580, K-41, SF-1195, M-4164A,A-32887, 30,504RP, 38,986, 44,161, 47,433, 47,434 and 47,224.

Each of the manganese complexes is produced and recovered by a processsimilar to that set forth in Example I except that the appropriatemicroorganism is utilized instead of the lasalocid producingmicroorganism. Specific processes for obtaining the named antibioticsare set forth above.

Some of the recovered manganese complex of each antibiotic is utilizedin a feed composition and fed to healthy and coccidiosis-infected groupsof food-producing animals such as cattle, sheep, swine and poultry whilethe remainder is purified in a process similar to that set forth inExample V. In accordance with the compositions and processes of thepresent invention, each of the manganese complexes, alone or incombination, is then administered to dogs to provide myocardialstimulation, or in treatment for coccidiosis in poultry, or for growthpromotion in food-producing animals such as cattle, sheep, swine andpoultry for growth promotion and as coccidiostats.

When fed to ruminants, the recovered manganese complex of eachantibiotic is formulated into a feed composition similar to thecomposition set forth in Example II and fed to cattle in amountssufficient to provide from about 5 to 100 ppm of the manganese complexin the rumen fluid during rumination. Positive effects are realized foreach polyether antibiotic in its manganese complexed form in promotinggrowth and feed efficiency in cattle. Similar results were also obtainedin myocardial stimulation in mammals.

While the present invention has been described it is understood thatnumerous modifications may be made by those skilled in the art withoutactually departing from the spirit and scope of the invention.

What is claimed is:
 1. A manganese complex of lysocellin.
 2. A feedadditive composition for food-producing animals comprising a carriermaterial and at least about 5 percent by weight on a dry basis of amanganese complex of lysocellin.
 3. A feed additive composition forfood-producing animals in accordance with claim 2 wherein the manganesecomplex of lysocellin comprises about 10 per cent to 50 per cent byweight on a dry basis of the composition, the composition furtherincluding said carrier material.
 4. A feed composition comprising anutrient feed material and a manganese complex of lysocellin.
 5. A feedcomposition in accordance with claim 4 comprising said nutrient feedmaterial and about 50 to 200 grams per ton of said manganese complex oflysocellin.
 6. A feed composition in accordance with claim 5 comprisingsaid nutrient feed material and about 75 to about 125 grams per ton ofsaid manganese complex of lysocellin.
 7. A process for promoting growthand enhancing feeding efficiency in food-producing animals which processcomprises administering to said food-producing animals agrowth-promoting and feed efficiency-enhancing amount of manganesecomplex of lysocellin.
 8. The process of claim 7, in which a biomasscontaining the manganese complex of lysocellin is fed to said animals,said biomass being prepared by:(a) fermenting a fermentation brothinoculated with a "Streptomyces" microorganism capable of producinglysocellin by fermentaton of the broth for a period of time and undersuitable fermentation conditions in order to produce said lysocellin insaid fermentation broth; (b) providing in said lysocellin-containingfermentation broth a water-soluble manganese salt in an amountsufficient to from a manganese complex of said lysocellin, which complexis insoluble in the fermentation broth; and (c) recovering said biomassof insoluble material from said fermentation broth, said biomasscontaining both a manganese complex of said lysocellin and insolublemanganese complexes of residual nitrogen-containing compounds present inthe fermentation broth.
 9. A process for promoting growth and enhancingfeed efficiency of food-producing animals in accordance with claim 7,which process comprises administering to food-producing animals a feedmaterial containing from about 50 to about 200 grams per ton of saidmanganese complex of lysocellin.
 10. A process in accordance with claim9 wherein said feed material contains from about 75 to about 125 gramsper ton of said manganese complex of lysocellin.
 11. An animal feedsupplement capable of increasing the rate of growth of an animal whensupplied in the animal's feed on a regular basis during the animal'snormal growth period, said animal feed supplement also being capable ofenhancing the feed efficiency of the animal, said feed supplementcomprising manganese lysocellin and a carrier material.
 12. A biomasscontaining a manganese complex of lysocellin, said biomass beingprepared by:(a) fermenting a fermentation broth inoculated with a"Streptomyces" microorganism capable of producing lysocellin byfermentation of the broth for a period of time and under suitablefermentation conditions in order to produce said lysocellin in saidfermentation broth; (b) providing in said lysocellin-containingfermentation broth a water-soluble manganese salt in an amountsufficient to form a manganese complex of said lysocellin, which complexis insoluble in the fermentation broth; and (c) recovering said biomassof said insoluble complex from said fermentation broth, said biomasscontaining both a manganese complex of said lysocellin and insolublemanganese complexes of residual nitrogen-containing compounds present inthe fermentation broth.